Field of the Invention
Embodiments of the subject matter disclosed herein relate generally to apparatuses, methods and systems for measurement of ion arrival time distribution and more particularly, to devices, processes, mechanisms and techniques for measuring size distribution and/or concentration of biological materials, such as protein molecules and includes other non-biological particles such as nanoparticles.
Description of Related Art
The ability to measure and to quantify the type, behavior, and/or characteristics of biomolecular ions (e.g., measurement of individual size and local number and/or mass concentrations) is of utmost importance in a large number of applications of interest, including, for example basic biological research, medical diagnostics, drug discovery, assessment of quality of biological substances and DNA sequencing. More specifically, in the medical diagnostics area, for example, there are many reasons to measure protein sizes and concentrations, including the ability to identify a genetic disease based on the variation of size and concentration of hemoglobin.
A variety of measurement techniques have been used to characterize biomolecular substances (e.g., DNA, RNA, and proteins, including their fragments). Gel electrophoresis, high pressure liquid chromatography, dynamic light scattering, capillary electrophoresis, mass spectrometry and ion mobility spectrometry are a some of the more important techniques. Gel electrophoresis is used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments, or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a gel matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. Gel electrophoresis can also be used for separation of nanoparticles. Those of ordinary skill will however recognize that gel electrophoresis is a technique that requires a substantial amount of time for completion of any given measurement. Additionally, the position of a band in a gel electrophoresis lane needs to be compared to size standards, typically molecules of known molecular weight, in order to estimate the molecular weight of the material in the band. The need to calibrate gel lanes adds to the effort involved and makes the method a relative measurement technique.
Modern chromatography instruments capably separate many types of complicated mixtures. By means of judicious choice of chromatography column packing material, many different types of biological molecules can be separated from each other so that lipids, proteins, peptides, hormones can be studied. Mass spectrometry provides a measure of an ion's mass. These instruments, while crucial to biological research, are expensive.
Differential electrical mobility analyzers may be used to determine the size distribution of biomolecular ions smaller than 100 nm. In this method, a cloud of charged aerosol particles is drawn between two electrodes, such as the annular space between two concentric cylinders. Voltage applied to the cylinders deflects particles of a predictable size into a particle detector. By scanning the voltage applied to the cylinders, the size distribution of the particles is obtained. U.S. Pat. No. 6,230,572 discloses an example of such an apparatus.
Another conventional device used to make measurements of ions is a drift tube 1, which is illustrated in FIG. 1A. In operation, voltage is applied to each of the ring-shaped electrodes 2 in such a way that the resulting electrical field inside the drift tube is constant along the longitudinal axis of the tube. A pulsed ion gate 3 is placed at the entrance to the drift tube and provides a way to introduce a pulse of ions 4 from an ion source, external to the drift tube, into the electric field generated inside the drift tube. In the example illustrated in FIG. 1A, the ion population is bimodal, i.e., it comprises a group of heavy ions 5 and another of light ions 6. An ion detector 7 is located at the opposite end of the drift tube and responds to ions when they strike the detector. In the exemplary illustration of FIG. 1A, the detector is a flat metal plate to which a current amplifier is connected and when a pulse of ions hits the detector, a momentary rise in detector current is observed, as illustrated in the Time-of-Flight (or TOF) spectrum 8 as shown in FIG. 1B for the bimodal ion group considered for this example. Ion drift tubes are commonly purged with a flow of background gas to minimize the influence of solvent vapor on drift time. Ion velocities resulting from the electrical field inside the drift tube are substantially higher than background gas velocities in the purge gas and, as a consequence, gas velocity has minimal influence on ion trajectories and does not significantly alter ion arrival time distributions. However, the performance of conventional drift tubes for massive ions having drift velocities close to the velocity of the purge gas is substantially degraded, as understood by those of ordinary skill in the applicable arts. Those skilled in the art will also appreciate the use of gas flowing countercurrent to the ions, which needs to be overcome by the ions.
Therefore, based at least on the above-noted challenges with conventional devices to measure the concentration and size of ions, it would be advantageous to have improved devices to accomplish the summarized tasks, among others, with increased measurement accuracy (particularly in embodiments operating on first principles without the need for calibration), lower cost of manufacturing and operation, reduction on the time required for measurements, and minimization or elimination of the effect of purge gas velocity on the velocity of the ions being measured, while, in some embodiments of the subject matter disclosed herein, increasing the resolution of such measurements by mathematically deconvolving from the measurements the effect of a spread in arrival times due to diffusion and non-ideal background flow velocity variations.